.alpha.-Glycerophosphate oxidase and useful techniques for its preparation and extraction have been described by Koditschek, L. K. and Umbreit, W. W., ".alpha.-Glycerophosphate Oxidase in Streptococcus faecium F24", Journal of Bacteriology, Vol. 98, No. 3, P. 1063-68 (1969) and by Jacobs, N. J. and Van Demark, P. J., "The Purification and Properties of the .alpha.-Glycerophosphate Oxidizing Enzyme of Streptococcus faecalis, 10Cl," Archives of Biochemistry and Biophysics, Vol. 88, p. 250-55 (1960). The enzyme is useful for the oxidiation of .alpha.-glycerophosphate in the presence of oxygen to produce dihydroxyacetone phosphate and hydrogen peroxide.
Koditschek and Umbreit, supra, described the properties of .alpha.-glycerophosphate oxidase in Streptococcus faecium F24 using for their study various assay techniques including the manometric assay. Cultures of Streptococcus faecium were maintained on stabs of AC agar consisting of 1% tryptone, 1% yeast extract, 0.5% K.sub.2 HPO.sub.4,0.1% g glucose and 1.5% agar (sometimes referred to as AC medium below). The cells were separated from this medium and enzyme preparations were extracted for assay.
Jacobs and Van Demark, supra, who also used manometric assay techniques in their study, describe the properties of .alpha.-glycerophosphate oxidase in Streptococcus faecalis 10Cl. They grew the Streptococcus faecalis in an AC medium as described above but having 0.2% glucose. They report that the enzyme from Streptococcus faecalis is highly specific for L-.alpha.-glycerophosphate as a substrate with no demonstrable activity on .beta.-glycerophosphate, glycerol, dihydroxyacetone phosphate, or 1,2-propanediol phosphate.
Jacobs, N. J. and Van Demark, P. J. in an article "Comparison of the Mechanism of Glycerol Oxidation in Aerobically and Anaerobically Grown Streptococcus faecalis", Journal of Bacteriology, Vol. 79, pp. 532-538 (1960) discuss the metabolism of glycerol in taxonomic studies using aerobically and anaerobically grown cells from Streptococcus faecalis. The Streptococcus faecalis was grown in the standard AC medium described above.
Gunsalus, I. C., and Umbreit, W. W., "The Oxidation of Glycerol by Streptococcus Faecalis", Journal of Bacteriology, 49, 347-357 (1945) report on assay studies of the oxidation of glycerol by active cell suspensions of Streptococcus faecalis. They found that the addition of pyruvate to the assay would allow the oxidation of higher levels of glycerol because pyruvate acts as a scavenger for H.sub.2 O.sub.2. .alpha.-Glycerophosphate oxidase activity was not noted.
Gunsalus, I. C., "Products of Anaerobic Glycerol Fermentation by Streptococcus Faecalis", Journal of Bacteriology, 54, 239-244 (1947) reports on the growth of Streptococcus faecalis in a medium containing glycerol and up to 1% yeast extract. Gunsalus describes the growth of Streptococcus faecalis with glycerol as slight (aerobic growth) or poor (anaerobic growth).
Claridge, C. A. and Hendlin, D., "Oxidation of Glycerol by Streptococcus Faecalis", Journal of Bacteriology, Vol. 84, p. 1181-86 (1962) describe studies of the utilization of glycerol by Streptococcus faecalis made using conventional warburg assays. They suggest growing Streptococcus faecalis in a medium containing 1 g/liter glucose supplemented with 1% glycerol (10 g/liter) in order to obtain cells that will rapidly oxidize glycerol in their assay. .alpha.-Glycerophosphate oxidase activity was not noted.
The references discussed above generally describe studies relating to the oxidation of glycerol. Only the first two of these references, "Koditschek and Umbreit" and "Jacobs and Van Demark", indicate the presence of .alpha.-glycerophosphate oxidase in their assays. Neither suggest that growing bacterium on a combination of (1) pyruvate, (2) an inducer for .alpha.-glycerophosphate oxidase and, preferably, (3) glucose will produce unexpectedly high yields of .alpha.-glycerophosphate oxidase. None of the other references even suggest the presence of .alpha.-glycerophosphate oxidase in their strains of microorganisms.